High-throughput screening instruments are critical tools in the pharmaceutical research industry and in the process of discovering and developing new drugs. The drug discovery process involves synthesis and testing, or screening, of candidate drug compounds against a target. A candidate drug compound is a molecule that might mediate a disease by its effect on a target. A target is a biological molecule, such as an enzyme, receptor, other protein, or nucleic acid, that is believed to play a role in the onset or progression of a disease or a symptom of a disease. FIG. 1 shows stages of the drug discovery process, which include target identification, compound synthesis, assay development, screening, secondary screening of hits, and lead compound screening, or optimization, and finally clinical evaluation.
Targets are identified based on their anticipated role in the progression or prevention of a disease. Until recently, scientists using conventional methods had identified only a few hundred targets, many of which have not been comprehensively screened. Recent developments in molecular biology and genomics have led to a dramatic increase in the number of targets available for drug discovery research.
After a target is selected, a library of compounds is selected to screen against the target. Compounds historically have been obtained from natural sources or synthesized one at a time. Compound libraries were compiled over decades by pharmaceutical companies using conventional synthesis techniques. Recent advances in combinatorial chemistry and other chemical synthesis techniques, as well as licensing arrangements, have enabled industrial and academic groups greatly to increase the supply and diversity of compounds available for screening against targets. As a result, many researchers are gaining access to libraries of hundreds of thousands of compounds in months rather than years.
Following selection of a target and compound library, the compounds must be screened to determine their effect on the target, if any. A compound that has an effect on the target is defined as a hit. A greater number of compounds screened against a given target results in a higher statistical probability that a hit will be identified.
Prior to screening compounds against a target, a biological test or assay must be developed. An assay is a combination of reagents that is used to measure the effect of a compound on the activity of a target. Assay development involves selection and optimization of an assay that will measure performance of a compound against the selected target. Assays are broadly classified as either biochemical or cellular. Biochemical assays usually are performed with purified molecular targets, which generally have certain advantages, such as speed, convenience, simplicity, and specificity. Cellular assays are performed with living cells, which may sacrifice speed and simplicity, but which may provide more biologically relevant information. Researchers use both biochemical and cellular assays in drug discovery research. Both types of assays may use a variety of detection modalities, including absorbance, radioisotopic, chemiluminescence, and photoluminescence.
Assays are then run to identify promising compound candidates or hits. Once a compound is identified as a hit, a number of secondary screens are performed to evaluate its potency and specificity for the intended target. This cycle of repeated screening continues until a small number of lead compounds are selected. The lead compounds are optimized by further screening. Optimized lead compounds with the greatest therapeutic potential may be selected for clinical evaluation.
Due to the recent dramatic increase in the number of available compounds and targets, a bottleneck has resulted at the screening stage of the drug discovery process. Historically, screening has been a manual, time-consuming process. Screening significantly larger numbers of compounds against an increasing number of targets requires a system that can operate with a high degree of automation, analytical flexibility, and speed.
The most common high-throughput screening assay techniques utilize absorbance, radio-isotope labeling, photoluminescence (e.g., fluorescence) intensity, photoluminescence (e.g., fluorescence) polarization, and time resolved fluorescence (e.g., phos-fluorescence). There is a desire in the high-throughput screening industry to provide alternatives to assays that use radio-isotopes because: (1) radio-isotopes are relatively expensive and there is a question as to how well they will perform at low volumes, (2) they pose potential health hazards to workers, and (3) their disposal is problematic. In contrast, photoluminescence and chemiluminescence-based assays generally are attractive because they offer a sensitive assay read-out capability that is significantly less problematic for workers and much easier to dispose of than radio-isotopes.
The types of assays that are desired for high-throughput screening are evolving constantly. As new assays are developed in research labs, tested, and published in literature or presented at scientific conferences, new assays become popular and many become available commercially. New analytical equipment then is developed to support the most popular commercially available assays.
Current screening systems operate with various degrees of automation. Automation, from sample dispensing to data collection, enables round-the-clock operation, thereby increasing the screening rate. Automated high-throughput screening systems usually include combinations of assay analyzers, liquid handling systems, robotics, computers for data management, reagents and assay kits, and microplates.
Most analyzers in use today offer only a single assay modality or a limited set of modalities with non-optimum peirfomance. To perform assays using different detection modes, researchers generally must switch single-mode analyzers and reconfigure the high-throughput screening line. Altematively, researchers may set up the high-throughput screening line with multiple single-mode analyzers, which often results in critical space constraints.
Most analyzers used today are not designed specifically for high-throughput screening purposes. They are difficult and expensive to integrate into a high-throughput screening line. Even after the analyzer is integrated into the high-throughput screening line, there often are many problems, including increased probability of system failures, loss of data, time delays, and loss of costly compounds and reagents.
The proliferation of targets and compounds to be screened has given rise to the need to conserve reagents in order to reduce total screening costs. However, detection systems in use today generally are not designed to permit significant reduction in assay volume, as evidenced by the need to run microplates with completely filled wells and high reporter group concentrations in order to achieve acceptable performance. Many analyzers are not sensitive enough to read results based on these smaller volumes. Inadequate sensitivity may result in missed hits, limited research capabilities, increased costs of compounds, assays, and reagents, and lower throughput.
Ninety-six-well microplate formats have been and still are commonly used throughout the high-throughput screening industry. Some high-throughput screening labs are using 384- and 768-well plates, and some labs are experimenting with 1536-, 3456-, and 9600-well microplates. FIG. 2 shows a stack of overlapping microplates with various well densities. Plate 30 has 96 wells. Plate 32 has 384 wells. Plate 34 has 1536 wells. Plate 36 has 3456 wells. Plate 38 has 9600 wells. FIG. 2 illustrates the substantial difference in well dimensions and densities that may be used in high-throughput screening assays.
The need for flexibility to handle various microplate well densities presents significant challenges because reduction in sample volume may sacrifice sensitivity and because light interaction at sample boundary interfaces, including those defined by container walls, often contributes significant background noise, causing losses in the signal-to-noise ratio, as different container densities, capacities, and geometries are used.
In addition to differences in well density and capacity, well geometries are highly variable. Well side walls vary, For example, some side walls are horizontal, whereas others are slanted. Well bottoms also vary. For example, some well bottoms are flat, whereas others are V-shaped or U-shaped. Well peripheral shapes also vary. For example, some well peripheral shapes are round, whereas others are square or hexagonal. Some wells have baffles for mixing or for increasing surface area. Each of these variations presents different potential interfacial boundary interference problems.
The problem of handling different sample volumes and container geometries is further complicated by other high-throughput screening objectives, such as the goal of offering multi-mode detection and assay variability. Interference from light interaction at interfacial boundaries around the sample may cause more of a problem for one type of assay compared to another. Consequently, inability to avoid, or at least manage, interference due to variations in container density, shape, and size inevitably imposes limitations on the assay flexibility of a given analyzer. It may be necessary to use sample containers of standard dimension, and it may be necessary to provide different analyzers for different modes of analysis.
Thus, prior detection devices have not generally recognized the need to provide analytic flexibility and high peiformance for assay development as well as ease of use and smooth automation interface for the high-throughput screening lab. A real need exists for a sensitive, versatile, multi-mode analyzer with high-throughput capability that can handle wide ranges of sample volumes and variations in container material, geometry, size and density format while reliably maintaining a high level of sensitivity.